Application Note: 407

Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS

1

Ting Liu1, Peter Wang1, and Kefei Wang2 Thermo Fisher Scientic, Shanghai, China; 2Thermo Fisher Scientic, San Jose, CA, USA

Introduction
Key Words TSQ Quantum Access Accela High Speed LC System Antibiotic Food Residue Analysis SRM Chloramphenicol (CAP) is a broad-spectrum antibiotic with historical veterinary uses in all major food-producing animals (see Figure 1 for structure). It has serious side effects on humans that may cause aplastic anemia, and the suspected carcinogen effect is also thought to be dose independent. Consequently, chloramphenicol has been banned for use in all food-producing animals by the European Union (EU), USA and Canada. A minimum required performance limit (MRPL) for chloramphenicol determination was recently set by the EU at 0.3 g/kg (ppb) in all foods of animal origin, such as meat, seafood, egg, milk, honey, etc. However, residues of CAP at unacceptable levels continue to be found in food imports, as a result of illegal use in some countries to mask the poor hygiene conditions of animal-raising farm and to augment animal growth. The growing food safety concerns call for intensive surveillance of chloramphenicol in food products.

Goal
To develop a simple, rapid, and sensitive LC-MS/MS method for analyzing chloramphenicol in milk. The method should be suitable for both screening and conrmatory purposes.

Experimental Conditions
Sample Preparation Standards and Regents: Chloramphenicol (98%) was purchased from Sigma-Aldrich (St. Louis, MO) and d5chloramphenicol (100 g/mL in acetonitrile) as internal standard from Cambridge Iosotope Lab (Andover, MA). Regent grade water, acetonitrile and methanol were from Thermo Fisher Scientic (Pittsburgh, PA).
Procedures:
0.5 g Milk + d5CAP (0.3 ppb) as IS

+ 0.75 mL CH3CN, vortex 1 min, Centrifuge @ 14000 rpm for 10 min

Take 0.7 mL Supernatant + 0.3 mL Water, store at 4C for 1 hr

Figure 1: Structure of chloramphenicol

Analysis of residual of chloramphenicol in foodstuff is challenging because of the complicated sample matrices and stringent requirements of both low quantitation limit (<0.3 ppb) and method validation. The technique of liquid chromatography separation followed by tandem mass spectrometry detection, LC-MS/MS, is the technology of choice because of its sensitivity and specicity. A sample cleanup process is generally required to remove the sample matrix prior to the LC-MS/MS run. Typically, this involves the costly and labor-intensive solid phase extraction (SPE) and/or liquid-liquid extraction (LLE) procedures. In this work, we report a simple sample preparation procedure involving only the acetonitrile protein precipitation and dilution to extract the CAP from milk, followed by a high-speed LC separation and detection by a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. The sample preparation is simple, fast, and inexpensive, and the method exceeds the sensitivity and specicity requirements for both screening and conrmatory assays. Validation according to the European Commission Decision 2002/657/EC has also been performed.

Pipette 0.8 mL upper solution for LC-MS/MS Analysis

Chromatography Conditions HPLC Module: Accela High Speed LC System (Thermo Scientic, San Jose, CA) Column: Hypersil GOLD 50 mm 2.1 mm and 1.9 m particle size (Thermo Scientic, Bellefonte, PA) Column Temperature: Ambient Mobile Phase: A: Methanol B: Water Gradient: Time (min) A% 0.0-0.6 5% 2.3 100% 2.35-3.0 5% Flow Rate: 500 L/min Injection Volume: 20 L (with loop)

Mass Spectrometer Conditions Mass Spectrometer: TSQ Quantum Access triple stage quadrupole mass spectrometer (Thermo Fisher Scientic, San Jose, CA) Source: ESI-, 3000 V Sheath Gas: 45 unit Auxiliary Gas: 10 unit Capillary Temperature: 300C Source CID: -7 V Q1 and Q3 Peak Width (FWHM): 0.7 Da Scan Time: 0.1 s Collision Gas: Ar (1.5 mTorr) SRM Transitions: 3 SRMs for CAP, 1 SRM for d5-CAP (see Table 1)
Product Ion (Collision Energy) 152 (17)* 257 (15) 194 (16) 157 (17)*

The results of relative ion abundance measured at various concentrations are given Table 2. Both relative ion abundance ratios of 257/152 and 194/152 meet the requirements set by Decision 2002/657/EC. Note that we found the 321>257 transition is more likely subjected to matrix interferences in many other cases of different matrices, thus if two SRM transitions need to be selected (4.0 IPs) for the method, 321>152 and 321>194 are preferred. Method Performance: Figure 2 shows representative SRM chromatograms for a blank and 0.05 g/kg spiked milk samples. As shown, with high-speed LC, each chromatographic run is only 3 min, allowing high throughput for screening assay. All three SRM traces for CAP at 0.05 g/kg spiked samples can be well quantied. Note that the 0.05 g/kg spiked in milk is equivalent to 0.46 pg injected on column by assuming a full recovery. It should also be noted that with the high-speed LC separation of only 3 min for each chromatographic run, the CAP peak width (at 10% above baseline) is as narrow as 6 s. Under current MS acquisition conditions, there are 13-14 points across each peak, enough for maintaining a well-dened peak shape for accurate integration. A representative calibration curve from standards prepared in milk is shown in Figure 3. Good linearity from 0.05 to 1.0 g/kg with correlation coefcient of R2= 0.9954 (Weighting factor W = 1/X) was obtained. Table 3 shows excellent recovery and within-laboratory reproducibility of the method (at four different days). Decision Limit (CC) and Detection Capability (CC): According to Decision 2002/657/EC, the Decision Limit CC is the minimum CAP concentration at which a sample is really non-compliant with an error probability of 1% ( =0.01), and the Detection Capability (CC) is the minimum amount of CAP that can be quantied and conrmed with an error probability of 5% ( =0.05). Two methods can be used for calculating the CC according to the Decision. One is to use the S/N ratio of 3:1 of blank samples, similar to those for estimation of limit of detection. The other is to use the intercept of calibration curve at low levels and the within-laboratory reproducibility. The former method does not work well for LC-MS/MS because the very low background (noise count ~0) of SRM chromatogram often yields unrealistically low values for CC. Thus we use the latter approach by using cali-

Precursor Ion

CAP (M -H -)

320.93

d 5 -CAP (M -H -)
* Product ion used for quantitation

326.93

Table 1: SRM transitions for CAP and d5-CAP (IS)

Results and Discussion

Sample Preparation: A major goal for the method development in this study is to avoid using the labor intensive and time-consuming SPE or LLE procedures as in literatures. In curret work, the proteins from milk were removed with acetonitrile precipitation at ratio of 1.5:1 (v/v Acetonitrile:Milk), followed by dilution with water, which is necessary for gradient chromatographic separation. At such ratio, protein removal was not complete, trace amount of precipitates of proteins appeared after the sample was stored at 4C for some time. Thus, the supernatant was taken for LC-MS/MS analysis after the sample was stored at 4C for 1 hr. Choice of Quantitation and Qualication Ions: Three product ions were chosen to give an Identication Points (IPs) of 5.5 to meet the requirement of 4.0 IPs by the Decision 2002/657/EC for conrmatory assay of the prohibited substances such as CAP. The m/z 152 was chosen as quantitation ion, the m/z 257 and 194 as conrmation ions, consisting with those reported in literatures.

CAP Spiked Level (g/kg) 0.05 0.15 0.30 0.50

Relative Ion Abundance of 257/152 Mean n= 6 96% 92% 93% 90% %RSD n= 6 16% 7.6% 15% 3.4% 20% Tolerance by Decision 2002/657/EC

Relative Ion Abundance of 194/152 Mean n= 6 26% 28% 31% 31% %RSD n= 6 21% 25% 15% 17% 25% Tolerance by Decision 2002/657/EC

Note: Relative ion abundance values were calculated by relative peak area ratios

Table 2: Relative ion abundances at various CAP concentrations in milk and the associated tolerances required by Decision 2002/657/EC

bration data of (0.05-0.15-0.30 g/kg) to obtain the Y-intercept and its standard deviation, SDY-intercept,
CC=Y-intercept + 2.33*SDY-intercept

with the within-laboratory reproducibility data of 0.15 g/kg spiking level, thus,
CC =CC + 1.64*SD0.15 g/kg

Similarly, the CC can be calculated from CC and the standard deviation of 20 measurement of samples spiked at CC level. Here the latter term is approximated

Where SD0.15 g/kg is the within-laboratory reproducibility (in standard deviation) of the 0.15 g/kg in Table 3. The calculated values of CC and CC are 0.087 g/kg and 0.12 g/kg, respectively.

CAP Spiking Level (g/kg) 0.05 0.15 0.30 0.50 Mean (%) 97% 101% 104% 94%

Within-laboratory Reproducibility (n = 20) SD (g/kg) 0.0065 0.020 0.037 0.042 %RSD 14% 13% 11% 8.0%

Table 3: Recovery and Reproducibility Data

Figure 2: SRM chromatograms for milk blank and 0.050 g/kg spiked milk samples

CAP Y = 0.148836 + 2.55752*X R^2 = 0.9954 W: 1/X 3.5 3.0 2.5

Area Ratio

2.0 1.5 1.0 0.5 0.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

g/kg
Figure 3: Calibration of CAP in milk

Conclusions
A simple, rapid and sensitive method for analysis of CAP in milk by LC-MS-MS has been developed and validated. The sample preparation by protein precipitation and dilution is very simple to perform and avoids the use of SPE or LLE. With the high-speed Accela LC coupled to a triple quadruple TSQ Quantum Access, each analytical run is as short as 3 min. The method can be used for the purposes of both high-throughput screening and rapid conrmatory assays. For screening assay, the method can detect < 0.050 g/kg CAP in milk. For conrmatory assay, the method validated according to Decision 2002/657/EC gives a CC =0.087 g/kg and CC = 0.12 g/kg, both below the MRPL of 0.3 g/kg.
References
1. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/ECD concerning the performance of analytical methods and the interpretation of results, Ofcial Journal of the European Communities, L 221, 2002, 8-36. 2. Bogusz, M.J. et al. Rapid determination of chloramphenicol and its glucuronide in food products by liquid chromatographyelectrospray negative ionization tandem mass spectrometry; J, Chrom. B 2004, 807(2), 343-356. 3. Tao, D. et al. Effects of sample preparation and high resolution SRM on LC-MS-MS determination of chloramphenicol in various food products; Poster Presentation, 53rd ASMS Conference, San Antonio, TX, USA, June 5-9, 2005. 4. Gallo, P. et al. Development of a liquid chromatography/electrospray tandem mass spectrometry method for conrmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein afnity chromatography; Rapid Commun. Mass Spectrom. 2005, 19(4), 574-579. 5. Vinci, F. et al. In-house validation of liquid chromatography electrospray tandem mass spectrometry method for conrmation of chloramphenicol residues in muscles according to Decision 2002/567/EC; Rapid Commun. Mass Spectrom. 2005, 19(22), 3349-3355.

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